1. Field of the Invention
This invention relates to the field of therapeutic peptides for the prevention and treatment of disorders or diseases resulting from abnormal formation of amyloid or amyloid-like deposits, such as, but not limited to, prion-related encephalophathies, Alzheimer's dementia or disease (AD), and other amyloidosis disorders. This invention also relates to the use of the peptides in preventing the formation of or in promoting the redissolution of these insoluble amyloid or amyloid-like deposits.
2. Description of the Background Art
Alzheimer's disease (AD) is the most common form of dementia in adults (C. Soto et al. J. Neurochem. 63:1191-1198, 1994), constituting the fourth leading cause of death in the United States. Approximately 10% of the population over 65 years old is affected by this progressive degenerative disorder that is characterized by memory loss, confusion and a variety of cognitive disabilities. One of the key events in AD is the deposition of amyloid as insoluble fibrous masses (amyloidogenesis) resulting in extracellular neuritic plaques and deposits around the walls of cerebral blood vessels. The main component of amyloid is a 4.1-4.3 kDa hydrophobic peptide, named amyloid .beta.-peptide (A.beta.), that is codified in chromosome 21 as part of a much longer amyloid precursor protein APP (Muller-Hill and Beyreuther, Ann. Rev. Biochem. 38:287-307, 1989). The APP starts with a leader sequence (signal peptide), followed by a cysteine-rich region, an acidic-rich domain, a protease inhibitor motif, a putative N-glycosylated region, a transmembrane domain, and finally a small cytoplasmic region. The A.beta. sequence begins close to the membrane on the extracellular side and ends within the membrane. Two-thirds of A.beta. faces the extracellular space, and the other third is embedded in the membrane (Kang et al. Nature 325:503-507, 1987; Dyrks et al. EMBO J. 7:949-957, 1988). Several lines of evidence suggest that amyloid may play a central role in the early pathogenesis of AD.
Evidence that amyloid may play an important role in the early pathogenesis of AD comes primarily from studies of individuals affected by the familial form of AD (FAD) or by Down's syndrome. Down's syndrome patients have three copies of APP gene and develop AD neuropathology at an early age (Wisniewski et al., Ann. Neurol. 17:278-282, 1985). Genetic analysis of families with hereditary AD revealed mutations in chromosome 21, near or within the A.beta. sequence (Forsell et al., Neurosci. Lett. 184:90-93, 1995). Moreover, recently it was reported that transgenic mice expressing high levels of human mutant APP progressively develop amyloidosis in brain (Games et al., Nature 373:523-527, 1995). These findings appear to implicate amyloidogenesis in the pathophysiology of AD.
Recently, the same peptide that forms amyloid deposits in AD brain was also found in a soluble form (sA.beta.) normally circulating in the human body fluids (Seubert et al., Nature 359:355-327, 1992; Shoji et al., Science 258:126-129, 1992). It is believed that the conversion of sA.beta. to insoluble fibrils is initiated by a conformational or proteolytic modification of the 2-3 amino acid longer soluble form. It has been suggested that the amyloid formation is a nucleation-dependent phenomena in which the initial insoluble "seed" allows the selective deposition of amyloid (Jarrett et al., Biochem. 32:4693-4697, 1993).
Peptides containing the sequence 1-40 or 1-42 of A.beta. and shorter derivatives can form amyloid-like fibrils in the absence of other protein (Pike et al., J. Neurosci. 13:1676-1687, 1993), suggesting that the potential to form amyloid resides mainly in the structure of A.beta.. The relation between the primary structure of A.beta. and its ability to form amyloid-like fibrils was analyzed by altering the sequence of the peptide. Substitution of hydrophilic residues for hydrophobic ones in the internal A.beta. hydrophobic regions (amino acids 17-21) impaired fibril formation (Lorenzo et al., Proc. Natl. Acad. Sci. USA 91:12243-12247, 1994), suggesting that A.beta. assembly is partially driven by hydrophobic interactions. Indeed, larger A.beta. peptides (A.beta.1-42/43) comprising two or three additional hydrophobic C-terminal residues are more amyloidogenic (Soto et al., J. Neurochem. 63:1191-1198, 1994). Secondly, the conformation adopted by A.beta. peptides is crucial in amyloid formation. A.beta. incubated at different pH, concentrations and solvents has mainly an .alpha.-helical (random coil) or a .beta.-sheet secondary structure (Hilbich et al., J. Mol. Biol. 228:460-473, 1992; Jarrett et al., Biochem 32:4693-4697, 1993; Barrow et al., J. Mol. Biol. 225:1075-1093, 1992). The A.beta. peptide with .alpha.-helical or random coil structure aggregates slowly; A.beta. with .beta.-sheet conformation aggregates rapidly (Burdick et al., J. Biol. Chem. 267:546-554, 1992; Zagorski et al., Biochem. 31:5621-5631, 1992; Soto et al., J. Biol. Chem. 270:3063-3067, 1995). The importance of hydrophobicity and .beta.-sheet secondary structure on amyloid formation also is suggested by comparison of the sequence of other amyloidogenic proteins.
Analysis of A.beta. aggregation by turbidity measurements indicates that the length of the C-terminal domain of A.beta. influences the rate of A.beta. assembly by accelerating nucleus formation (Soto et al., 1994, supra; Soto et al, Neurosci. Lett. 186:115-118, 1995). Thus, the C-terminal domain of A.beta. may regulate fibrillogenesis. However, in vitro modulators of A.beta. amyloid formation such as metal cations (Zn, Al) (Soto et al., Biochem. J. 314:701-707, 1996; Jarrett et al., Cell 73:1055-058, 1993), heparan sulphate proteoglycans (Bush et al., Science 265:1464-1467, 1994) and apoliprotein E (Exley et al., FEBS Lett. 324:293-295, 1993) interact with the 12-28 region of A.beta.. Moreover, mutations in the .beta.PP gene within the N-terminal A.beta. domain yield analogs more fibrillogenic (Soto et al., 1995, supra; Buee et al., Brain Res. 627:199-204, 1993; Strittmatter et al., Proc. Natl. Acad. Science. (USA) 90:1977-1981, 1993; Wisniewski et al., Biochem. Biophys. Res. Commun. 179:1247-1254, 1991). Finally, while the C-terminal domain of A.beta. invariably adopts a .beta.-strand structure in aqueous solutions, environmental parameters determine the existence of alternative conformation in the A.beta. N-terminal domain (Hilbich et al., 1992, supra; Burdick et al., 1992, supra). Therefore, the N-terminus may be a potential target site for inhibition of the initial random coil to .beta.-sheet conformational change.
The emerging picture from studies with synthetic peptides is that A.beta. amyloid formation is dependent on hydrophobic interactions of A.beta. peptides adopting an antiparallel .beta.-sheet conformation and that both the N- and C-terminal domains are important for amyloid formation. The basic unit of fibril formation appears to be the conformer adopting an antiparallel .beta.-sheet composed of strands involving the regions 10-24 and 29-40/42 of the peptide (Pike et al., 1993, supra; Clements et al., Neurosci. Lett. 161:17-20, 1993). Amyloid formation proceeds by intermolecular interactions between the .beta.-strands of several monomers to form an oligomeric .beta.-sheet structure precursor of the fibrillar .beta.-cross conformation. Wood et al., 1995, supra, reported the inserting of aggregation-blocking prolines into proteins and peptides to prevent aggregation without affecting the structure or function of the native protein. In this manner, the authors suggest that novel proteins can be designed to avoid the problem of aggregation as a barrier to their production.
To date there is no cure or treatment for AD and even the unequivocal diagnosis of AD can only be made after postmortem examination of brain tissues for the hallmark neurofibrillary tangles (NFT) and neuritic plaques. However, there are several recent publications outlining strategies for the treatment of Alzheimer's disease.
Heparin sulfate (glycosoaminoglycan) or the heparin sulfate proteoglycan, perlecan, has been identified as a component of all amyloids and has also been implicated in the earliest stages of inflammation-associated amyloid induction. Kisilevsky et al., Nature Medicine 1(2):143-148, (1995) describes the use of low molecular weight (135-1,000 Da) anionic sulfonate or sulfate compounds that interfere with the interaction of heparin sulfate with the inflammation-associated amyloid precursor and the .beta.-peptide of AD. Heparin sulfate specifically influences the soluble amyloid precursor (SAA2) to adopt an increased .beta.-sheet structure characteristic of the protein-folding pattern of amyloids. These anionic sulfonate or sulfate compounds were shown to inhibit heparin-accelerated Alzheimer's A.beta. fibril formation and were able to disassemble preformed fibrils in vitro as monitored by electron micrography. Moreover, when administered orally at relatively high concentrations (20 or 50 mM), these compounds substantially arrested murine splenic inflammation-associated amyloid progression in vivo in acute and chronic models. However, the most potent compound, poly-(vinylsulfonate), was acutely toxic.
Anthracycline 4'-iodo-4'-deoxy-doxorubicin (IDOX) has been observed clinically to induce amyloid resorption in patients with immunoglobin light chain amyloidosis (AL). Merlini et al., Proc. Natl. Acad. Sci. USA 92:2959-2963 (1995), elucidated its mechanism of action. IDOX was found to bind strongly via hydrophobic interactions to two distinct binding sites (Scatchard analysis) in five different tested amyloid fibrils, inhibiting fibrillogenesis and the subsequent formation of amyloid deposits in vitro. Preincubation of IDOX with amyloid enhancing factor (AEF) also reduced the formation of amyloid deposits. Specific targeting of IDOX to amyloid deposits in vivo was confirmed in an acute murine model. This binding is distinct from heparin sulfate binding as removal of the glycosaminoglycans from extracted amyloid fibrils with heparinases did not modify IDOX binding. The common structural feature of all amyloids is a .beta.-pleated sheet conformation. However, IDOX does not bind native amyloid precursor light chains which suggests that the .beta.-pleated sheet backbone alone is not sufficient to form the optimal structure for IDOX binding, and that it is the fibril cross-.beta.-sheet quaternary structure that is required for maximal IDOX binding. It has been found that the amount of IDOX extracted from spleens is correlated with amyloid load and not circulating serum precursor amyloid levels. IDOX, however, is also extremely toxic.
The regulation and processing of amyloid precursor protein (APP) via inhibition or modulation of phosphorylation of APP control proteins has also been investigated in U.S. Pat. No. 5,385,915 and WO 9427603. Modulating proteolytic processing of APP to nucleating forms of AD has also been examined in AU 9338358 and EP569777. WO 95046477 discloses synthetic peptides of composition X-X-N-X coupled to a carrier, where X is a cationic amino acid and N is a neutral amino acid, which inhibit A.beta. binding to glycosoaminoglycan. Peptides containing Alzheimer's A.beta. sequences that inhibit the coupling of .beta.-1-antichymotrypsin and A.beta. are disclosed in WO 9203474.
Abnormal protein folding is also widely believed to be the cause of prion-related encephalophathies, such as Creutzfeldt-Jakob disease (CJD) and Gerstmann-Straussler-Scheinker disease (GSS) in humans, scrapie in sheep and goats, and spongiform encephalopathy in cattle.
The cellular prion protein (PrP.sup.c) is a sialoglycoprotein encoded by a gene that in humans is located on chromosome 20 (Oesch, B. et al., Cell 40:735-746, (1985); Basler, K. et al., 46:417-428 (1986); Liao, Y. J. et al., Science 233:364-367 (1986); Meyer, R. K. et al., Proc. Natl. Acad. Sci. USA 83:2310-2314 (1986); Sparkes, R. S. et al., Proc. Natl. Acad. Sci. USA 83:7358-7362 (1986); Bendheim, P. E. et al. J. Infect. Dis. 158:1198-1208 (1988); Turk, E. et al. Eur. J. Biochem. 176:21-30 (1988)). The PrP gene is expressed in neural and non-neural tissues, the highest concentration of mRNA being in neurons (Chesebro, B. et al., Nature 315:331-333 (1985); Kretzschmar, H. A. et al., Am. J. Pathol. 122:1-5 (1986); Brown, H. R. et al., Acta Neuropathol. 80:1-6 (1990); Cashman, N. R. et al., Cell 61:185-192 (1990); Bendheim, P. E., Neurology 42:149-156 (1992)).
The translation product of PrP gene consists of 253 amino acids in humans (Kretzschmar, H. A. et al., DNA 5:315-324 (1986); Pucket, C. et al., Am. J. Hum. 49:320-329 (1991)), 254 in hamster and mice or 256 amino acids in sheep and undergoes several post-translational modifications. In hamsters, a signal peptide of 22 amino acids is cleaved at the N-terminus, 23 amino acids are removed from the C-terminus on addition of a glycosyl phosphatidylinositol (GPI) anchor, and asparagine-linked oligosaccharides are attached to residues 181 and 197 in a loop formed by a disulfide bond (Turk, E. et al., Eur. J. Biochem. 176:21-30 (1988); Hope, J. et al., EMBO J. 5:2591-2597 (1986); Stahl, N. et al., Cell 51:229-240 (1987); Stahl, N. et al., Biochemistry 29:5405-5412 (1990); Safar, J. et al., Proc. Natl. Acad. Sci. USA 87:6377 (1990)).
In prion-related encephalopathies, PrP.sup.c is converted into an altered form designated PrP.sup.Sc, that is distinguishable from PrP.sup.c in that PrP.sup.Sc (1) aggregates; (2) is proteinase K resistant in that only the N-terminal 67 amino acids are removed by proteinase K digestion under conditions in which PrP.sup.c is completely degraded; and (3) has an alteration in protein conformation from .alpha.-helical for PrP.sup.Sc to an altered form (Oesch B. et al., Cell 40:735-746 (1985); Bolton, D. C. et al., Science 218:1309-1311 (1982); McKinley, M. P. et al., Cells 35:57-62 (1982); Bolton, D. C. et al., Biochemistry 23:5898-5905 (1984); Prusiner, S. B. et al., Cell 38:127-134 (1984); Bolton, D. C. et al., Arch. Biochem. Biophys. 258:1515-22 (1987)).
Several lines of evidence suggest that PrP.sup.Sc may be a key component of the transmissible agent responsible for prion-related encephalopathies (Prusiner, S. B. Science 252:1515-22 (1991)) and it has been established that its protease-resistant core is the major structural protein of amyloid-like fibrils that accumulate intracerebrally in some of these conditions (Brendheim, P. E. et al., Nature 310:418-421 (1984); DeArmond, S. J. et al., Cell 41:221-235 (1985); Kitamoto, T. et al., Ann. Neurol. 20:204-208 (1986); Robert, G. W. et al., N. Engl. Med. 315:1231-1233 (1986); Ghetti, B. et al., Neurology 39:1453-1461 (1989); Tagliavini, F. et al., EMBO J. 10:513-519 (1991); Kitamoto, T. et al., Neurology 41:306-310 (1991)).
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